1. Field of the Invention
This invention pertains to the field of in vitro sialylation of glycoproteins, including recombinant glycoproteins.
2. Background
The circulatory lifetime of glycoproteins in the blood is highly dependent on the composition and structure of its N-linked carbohydrate groups. This fact is of direct relevance for therapeutic glycoproteins which are intended to be administered parenterally. In general, maximal circulatory half life of a glycoprotein requires that its N-linked carbohydrate groups terminate in the sequence NeuAc-Gal-GlcNAc. Without the terminal sialic acid (NeuAc), the glycoprotein is rapidly cleared from the blood by a mechanism involving the recognition of the underlying N-acetylgalactosamine (GalNAc) or galactose (Gal) residues (Goochee et al. (1991) Biol/Technology 9: 1347-1355). For this reason, ensuring the presence of terminal sialic acid on N-linked carbohydrate groups of therapeutic glycoproteins is an important consideration for their commercial development.
In principle, mammalian cell culture systems used for production of most therapeutic glycoproteins have the capacity to produce glycoproteins with fully sialylated N-linked carbohydrate groups. In practice, however, optimal glycosylation is often difficult to achieve. Under the conditions of large scale production, overproduction of the glycoprotein by the cell can outstrip its ability to keep up with glycosylation, and this capability can be positively and negatively influenced by many subtle variables in culture conditions (Goochee et al., supra.).
Production of glycoproteins in transgenic animals has some of the same problems as mammalian cell culture. While the xe2x80x9cproductionxe2x80x9d of a glycoprotein is inherently better controlled, it is also less susceptible to manipulation. If glycosylation is not complete, there is little that can be done with the animals to alter the outcome. With transgenic animals there is often another problem. While the predominant sialic acid in humans is N-acetyl-neuraminic acid (NeuAc), goats, sheep and cows all produce a large fraction of their total sialic acid as N-glycolyl-neuraminic acid (NeuGc). Although the impact of this modification is not yet fully explored from a functional or regulatory perspective, it is known that the NeuGc substitution is antigenic in humans (Varki (1992) Glycobiology 2: 25-40).
Since the most important problems associated with glycosylation of commercially important recombinant and transgenic glycoproteins, involve terminal sialic acid, a need exists for an in vitro procedure to enzymatically xe2x80x9ccapxe2x80x9d carbohydrate chains that lack a terminal sialic acid. With such a procedure, the problem encountered with transgenic glycoproteins could also be addressed by resialylation with NeuAc once the xe2x80x9cnon-humanxe2x80x9d sialic acid NeuGc was removed. The ideal method would employ a sialyltransferase that is capable of efficiently sialylating N-linked or O-linked oligosaccharides of recombinant glycoproteins on a practical scale. The present invention fulfills these and other needs.
The present invention provides methods for in vitro sialylation of saccharide groups present on a recombinantly produced glycoprotein. The methods comprise contacting the saccharide groups with a sialyltransferase, a sialic acid donor moiety, and other reactants required for sialyltransferase activity for a sufficient time and under appropriate conditions to transfer sialic acid from the sialic acid donor moiety to said saccharide group.
In a preferred embodiment, the methods are carried out using sialyltransferase at a concentration of about 50 mU per mg of glycoprotein or less, preferably between about 5-25 mU per mg of glycoprotein. Typically, the concentration of sialyltransferase in the reaction mixture will be between about 10-50 mU/ml, with the glycoprotein concentration being at least about 2 mg/ml of reaction mixture. In a preferred embodiment, the method results in sialylation of greater than about 80% of terminal galactose residues present on said saccharide groups. Generally, the time required to obtain greater than about 80% sialylation is less than or equal to about 48 hours.
Sialyltransferases that are useful in the methods of the invention typically have a sialyl motif that comprises about 48-50 amino acids, within which about 40% of the amino acids are identical to the consensus sequence RCAVVSSAG - - - DVGSKT (SEQ ID NO:1)(where - - - indicates a variable number of amino acid residues such that the motif is about 48-50 residues in length). Examples of sialyltransferases that are suitable for use in the present invention include ST3Gal III (preferably a rat ST3Gal III), ST3Gal IV, ST3Gal I, ST6Gal I, ST3Gal V, ST6Gal II, ST6GalNAc I, ST6GalNAc II, and ST6GalNAc III (the sialyltransferase nomenclature used herein is as described in Tsuji et al. (1996) Glycobiology 6: v-xiv). The methods of the invention can involve sialylation of recombinant glycoproteins with more than one sialyltransferase; for example, with an ST3 Gal III and an ST3Gal I, or an ST3 Gal III and an ST6 Gal I, or other combinations of enzymes. The sialic acid donor moiety used in the claimed methods is generally CMP-sialic acid, which can be added to the reaction directly or can be enzymatically generated in situ. The sialic acids used in a preferred embodiment are selected from the group consisting of NeuAc and NeuGc.
The invention also provides a glycoprotein having an altered sialylation pattern, wherein terminal galactose residues of said glycoprotein are sialylated using the claimed methods.